3.7 Promotion of angiogenesis and proliferation in vivo
Angiogenesis is a complicated physiological process, playing an important role in wound healing (Potente et al. , 2011). In vivo , the observation of new vessels is a direct way to evaluate angiogenic activity. CD31 is a key biomarker of endothelial cells in blood vessels, and neovascularization was evaluated via immunofluorescence staining of CD31 in the cutaneous wounds. Newly formed vessels were detected in all groups, with higher abundance for FGF2 and compound 6 treatments (Figure 8A ). There was significantly better vessel density in the wound beds treated with compound 6 (57.67 ± 7.50 / mm2) than that in wounds treated with FGF2 (31.33 ± 4.16 / mm2) (Figure 8B ), suggesting compound 6 was more effective in promoting angiogenesis in the wound bed.
The pro-proliferation activity in vivo around the wound edge was further verified by immunohistochemistry staining of Proliferating cell nuclear antigen (PCNA). Consistent with the in vitro cellular proliferation result, there were few PCNA positive cells around the wound in PBS (control) mice. In contrast, an increased number of proliferating cells were detected in the epidermis and hair follicle for mice treated with compounds 5 , 6, and 7, or FGF2, with 1.5-fold more proliferating cells for compound 6treatment compared to treatment with FGF2 (Figure 8 C-D ).
All these data demonstrate that these FGF2 conjugates can effectively promote the proliferation and migration of epithelium and dermal cells, and enhance the activity of vascular regeneration, hence accelerating cutaneous wound healing (Figure 8E ). Compound 6exhibits the most potent wound healing with good stability.